PURIFICATION AND PROPERTIES OF ACID BETA-GALACTOSIDASE FROM FELINE LIVER

Acid β-galactosidase (EC 3.2.1.23) was purified 19 000-fold from feline liver with a 13% recovery using a four-step procedure that involved (1) lectin chromatography on concanavalin A-Sepharose 4B, (2) ion-exchange chromatography on DEAE-cellulose, (3) affinity chromatography on Sepharose 4B-6-aminohexyl 1-thio-β-d-galactopyranoside. and (4) gel filtration on Sepharose 6B. The purified protein eluted from Sepharose 6B as two symmetrical peaks of protein coincident with two peaks of enzyme activity. The two forms of the enzyme had apparent molecular weights of 700 000 and 130 000. Molecular weight estimation by sucrose gradient centrifugation revealed a single 115 000 molecular weight form. Reduced and denatured β-galactosidase migrated as a single major band with an apparent molecular weight of 62 000 during polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purified protein demonstrated a single protein precipitin arc that coincided with a single arc of enzyme activity when examined by immunoelectrophoresis, using an antiserum to partially purified feline liver acid β-galactosidase. Over 95% of the acid β-galactosidase activity from liver supernatants and the purified enzyme preparation was precipitated in immunotitration experiments. The amount of antiserum needed to precipitate a given quantity of activity was the same in both cases. Purified β-galactosidase hydrolyzed synthetic β-D-galactosides, α-L-arabinosides, and β-d-fucosides, as well as the β-linked galactose moieties of ganglioside GM1 and asialofetuin. The enzyme also catalyzed the transfer of galactose from p-nitrophenyl β-d-galactoside and ganglioside GM1 to various carbohydrate acceptors. © 1979, American Chemical Society. All rights reserved.